1. Field of the Invention
The present invention relates to the production of natural and modified forms of Factor IX. In particular, the invention relates to a transgenic animal containing, stably incorporated in its genomic DNA, an exogenous Factor IX gene that is expressed specifically in mammary tissue, such that Factor IX is secreted into milk produced by the animal. In particular, the invention relates to the production of human Factor IX in the milk of a transgenic non-human mammal using a DNA molecule that comprises a whey acidic protein promoter gene, 5xe2x80x2 regulatory sequences containing the promoter, human Factor IX cDNA that lacks at least a portion of the complete or any portion of or the complete the 3xe2x80x2-untranslated region of the native human Factor IX gene, but contains the 5xe2x80x2 and 3-untranslated region of the mouse whey acidic protein. gene.
2. Background
Human Factor IX, or xe2x80x9cChristmas factor,xe2x80x9d is encoded by a single-copy gene residing on the X-chromosome at q27.1. For a review of Factor IX gene structure and expression, see High et al., xe2x80x9cFactor IX,xe2x80x9d in MOLECULAR BASIS OF THROMBOSIS AND HEMOSTASIS, High (ed.), pages 215-237 (Dekker 1995); Kurachi et al., Thromb. Haemost. 73:333 (1995). The Factor IX gene is at least 34 kilobase (kb) pairs in size, and it is composed of eight exons. The major transcription start site of the Factor IX gene in human liver is located at about nucleotide-176. The human Factor IX mRNA is composed of 205 bases for the 5xe2x80x2 untranslated region, 1383 bases for the prepro Factor IX, a stop codon and 1392 bases for the 3xe2x80x2 untranslated region.
Factor IX is synthesized as a prepropolypetide chain composed of three domains: a signal peptide of 29 amino acids, a propeptide of 17 amino acids, which is required for xcex3-carboxylation of glutamic acid residues, and a mature Factor IX protein of 415 amino acid residues. The Factor IX zymogen undergoes three types of post-translational modifications before it is secreted into the blood: a vitamin K-dependent conversion of glutamic acid residues to carboxyglutamic acids, addition of hydrocarbon chains, and xcex2-hydroxylation of an aspartic acid. Mature Factor IX protein contains 12 xcex3-carboxylated glutamic acid (Gla) residues. Due to the requirement of vitamin K by xcex3-carboxylase, Factor IX is one of several vitamin K-dependent blood coagulation factors.
The activation of Factor IX is achieved by a two-step removal of the activation peptide (Ala146-Arg180) from the molecule. Bajaj et al., xe2x80x9cHuman factor IX and factor IXa,xe2x80x9d in METHODS IN ENZYMOLOGY (1993). The first cleavage is made at the Arg145-Ala146 site by either Factor XIa or Factor VIIa/tissue factor. The second, and rate limiting cleavage is made at Arg180-Val181. The activation pathways involving Factor XIa and Factor VIIa/tissue factor are both calcium-dependent. However, the Factor VIIa/tissue factor pathway requires tissue factor that is released from damaged endothelial cells. Activated human Factor IX thus exists as a disulfide linked heterodimer of the heavy chain and light chain. For full biological activity, human Factor IX must also have the propeptide removed and must be fully xcex3-carboxylated. Kurachi et al., Blood Coagulation and Fibrinolysis 4:953 (1993).
Factor IX is the precursor of a serine protease required for blood clotting by the intrinsic clotting pathway. Defects in Factor IX synthesis result in hemophilia B (or Christmas disease), an X-linked disorder that occurs in about one in 30,000 males. Patients with hemophilia B are treated with Factor IX obtained from pooled plasma from normal individuals. Martinowitz et al., Acta Haematol 94(Suppl. 1):35 (1995). Such Factor IX preparations, however, may be pyrogenic and may be contaminated with pathogenic agents or viruses. Accordingly, it would be advantageous to develop a means to prepare purified Factor IX that did not require extraction from human plasma.
In the past, therapeutic proteins have been produced in E. coli. However, limitations in secretion and post-translational modification which occur in all living cells has rendered recombinant protein production a highly species, tissue and cell specific phenomena. In an example of recombinant FIX expression in mammalian cells, the populations of recombinant FIX produced in baby hamster kidney cells are not the same protein products as FIX produced in Chinese hamster ovary cells (Busby et al., Nature 316:684-686 (1985); Kaufman et al., J. Biol. Chem. 261: 9622-9628 (1986)). These proteins have profound differences in xcex3-carboxylation and propeptide removal and these differences have been established as being very important in determining biological activity. Most importantly, only less than about 40 milliunits/hr/ml of active rFIX were detected in CHO cells even after coexpression of the propeptide cleaving enzyme PACE, coexpression of the carboxylase enzyme, and extensive gene amplification with methotrexate in an attempt to increase expression level and activity (Wasley et al. J. Biol. Chem. 268: 8458-8465 (1993); Rehemtulla et al., Proc. Natl. Acad. Sci. (USA), 90: 4611-4615 (1993)). Researchers concluded that multiple limitations in the secretion of active rFIX exist in mammalian cells (Rehemtulla et al., 1993) and that the problem of gene transcription was secondary and indeed trivial with respect to post-translational processing of biologically active rFIX in mammalian cells. Thus, FIX mRNA splicing is a species specific effect occurring in mice and perhaps sheep, but not pigs. Although one might hypothesize that a FIX could be expressed, one could not predict with any certainty whether such product would be a clinically acceptable, practical, recombinant therapeutic FIX product for a given hemophiliac indication.
Production of recombinant Factor IX in mammalian cell culture (HepG2, mouse fibroblast, mouse hepatoma, rat hepatoma, BHK, CHO cells) repeatedly has been shown to be recalcitrant and cell-system specific with respect to intracellular restrictions on secretion and proteolytic processing, post-translational modification, expression levels, biological activity, downstream recovery from production media, and substantiation of circulation half-life (Busby et al., (1985); de la Salle et al. Nature 316: 268-270 (1985); Anson et al., Nature 315: 684-686 (1985); Rehemtulla et al., 1993; Wasley, et al., (1993); Kaufman et al., (1986); Jallat et al., EMBO J. 9: 3295-3301 (1990)). Importantly, the aforementioned works concluded that nontrivial improvements in these combined criteria are needed if a practical prophylactic FIX therapeutic product is to be made available from any recombinant mammalian cell production source. For example, attempts to increase the specific activity of rFIX produced by CHO cells by rectifying problems with under-carboxylation by co-expression of the vitamin K-dependent carboxylase enzyme resulted in no improvement in xcex3-carboxylation or biological activity (Rehemtulla et al., (1993), implying that multiple rate limitations in this post-translational modification exist.
Similar difficulties in the production of significant amounts of biologically active rFIX in the mammary epithelial cells of transgenic animals also has been documented in the literature. Although WO-A-90/05188 and WO-A-91-08216 predict that production of rFIX should be possible in their production systems, no data are presented in WO-A-91-08216, and only very low levels of secreted rFIX (25 ng/ml) with no biological activity were reported in transgenic sheep in WO-A 90/05188 and in related publications (Clark et al., Bio/Technology 7: 487-4992 (1989)). Higher expression levels have recently been reported in the milk of sheep (5 xcexcg/ml), but again, the product had no biological activity (Colman, IBC Third International Symposium on Exploiting Transgenic Technology for Commercial Development, San Diego, Calif. (1995)). This demonstrates that the polypeptides produced in WO-A-90/05188, Clark et al. (1989), and Colman (1995) were a different species than native human FIX with dissimilar biological activity to human FIX, and could never be used for therapeutic purposes. Work by Clark et al. (1992) stated that problems in synthesis of rFIX in the mammary gland of transgenic mice was the result of aberrant splicing of the rFIX mRNA in the 3xe2x80x2 untranslated region. Correction of the aberrant splicing in transgenic mice has been demonstrated (Yull et al. Proc. Natl. Acad. Sci. USA 92: 10899-10903 (1995); Clark, et al. (1989), WO 95/30000)), resulting in higher expression levels (up to 61 xcexcg/ml) with about 40% biological active material. However, this aberrant splicing phenomenon appears to be species- and tissue-specific in the mouse mammary gland; other reports with the 3xe2x80x2 UTR sequences in CHO cell lines and in the liver of transgenic mice specifically show no evidence of aberrant splicing (Kaufman et al., (1986); Jallat et al., (1990)). In addition, no evidence was reported for aberrant mRNA splicing of FIX transcripts with 3xe2x80x2 UTR sequences in a human hepatoma cell line (de la Salle et al., (1985)), a mouse fibroblast cell line (de la Salle et al., (1985)), a rat hepatoma cell line (Anson et al., 1985)), or a BHK cell line (Busby et al., (1985)). No data are presented to justify the prediction that the altered transgene of WO95/30000 will necessarily improve the secretion and biological activity of rFIX in the milk of transgenic livestock or any other cell line. Therefore the claims presented in WO 95/30000 are purely speculative and are limited to the mammary gland of transgenic mice.
The stability of the rFIX product in the milk of transgenic livestock during upstream and downstream processing is a critical issue for the production of a practical therapeutic. Data presented in Clark et al. (1989) showed that Clark""s method of downstream recovery of what little rFIX was in the milk of their transgenic sheep was not reproducible: in one of the preparations, a significant amount of rFIX was proteolytically activated. The infusion of activated FIX (FIXa) into a patient is fatal (Kingdon et al., Thrombosis, Diathes. Haemorrh. (Stuttg.) 33: 617 (1975)). FIX can be activated by FXI and/or FVIIa/Tissue factor complex in the presence of calcium and phospholipids (Kurachi et al., Blood Coagulation and Fibrinolysis 4: 953-974 (1993)). Milk is a medium containing calcium and phospholipid surfaces. In addition, there is extensively conserved homology between mammalian blood coagulation factors, especially between porcine FXI and human FXI (Mashiko and Takahashi, Biol. Chem. Hoppe-Seyler 375: 481-484 (1994)). Detectable levels of porcine FVII(a) and FXI(a) in the milk of nontransgenic pigs, and elevated levels of FVII(a) and FXI(a) in the milk of a pig with mastitic milk have been measured. Thus, one could predict that the recovery of a useful unactivated rFIX produced in the milk of transgenic livestock will be very sensitive to the health of the mammary gland (i.e., no subclinical or clinical mastitis), to the milking procedure (i.e., no tissue damage), to pretreatment of the milk immediately after collection, to storage of the milk before processing, and to the purification and formulation process itself. One would also predict that the undesirable in vivo activation of rFIX also can be minimized by the coexpression of inhibitors to FVIIa/TF such as the Tissue Factor Pathway Inhibitor (TFPI) protein, also called LACI, or the hybrid protein FX-LACI which is also a known inhibitor to FVIIa/TF. Although specific inhibitors of FXIa have not been identified, a similar approach can be made for neutralizing FXIa activation by coexpression of analogues of polypeptide substrates of FXIa similar to those that are commercially available for amidolytic assays. Yet another strategy may be to overexpress rFIX at very high levels ( greater than 1 g/l milk) such that the FIX activating enzyme is extremely limiting. Otherwise, steps must he taken immediately after milk collection to minimize activation. These include, but are not limited to, chelation of calcium (e.g., addition of EDTA), phospholipid removal, adjustment of pH, storage in ultra-low freezers, controlled thawing procedures, addition of protease inhibitors, and purification procedures that maintain minimal activation conduciveness. If activated rFIX still persists in the purified product, removal can be facilitated by lectin chromatography(N-linked carbohydrate moieties exist only in the activation peptide), immunoaffinity chromatography using a Mab directed to the activation peptide, or by metal ion induced precipitation techniques that can select for the differences in molecular stability of unactivated vs. activated FIX. Because of these inherent difficulties in production of active FIX at sufficiently high levels in mammalian cells and transgenic livestock, gene therapy has been cited as perhaps a more practical way of achieving a prophylactic therapeutic rather than recombinant technology (Kurachi et al., (1993); Kay et al., Proc. Natl. Acad. Sci. USA 91: 2353-2357 (1994)); Fallaux et al., Thromb-Haemost. 74: 266-73 (1995)). This is certainly a profound reality because it specifically teaches a product suitable for FIX prophylaxis has not yet been found using recombinant production in mammalian cells, even those that have been shown to express active FIX, albeit at low levels. The best recombinant FIX cell production system made from CHO cells is produced at low secretion levels (Rehemtulla et al., (1993)) and is in fact not suitable for prophylaxis. Furthermore, the data have shown that the homologous plasma proteins FIX and protein C all have very different, cell-specific restrictions on post-translational processing, proteolytic processing, and secretion which preclude on a protein-specific basis the predictability of high expression levels, biological activity, downstream recovery from production media, and predictable circulation half-life (Grinnell et al., xe2x80x9cNative and Modified recombinant human protein C: function, secretion, and postranslational modifications,xe2x80x9d In Protein C and Related Anticoagulants, eds. D. F. Bruley and Drohan 29-63, Gulf Publishing Co., Houston, Tex. (1990); Yan et al., Trends in Biochem. Sci. (1989); Busby et al., (1985)).
Therefore, a need still exists for a means to obtain significant amounts of purified Factor IX from a source other than human plasma. A need also exists for a practical means for producing in mammalian cells rFIX, which is suitable as a treatment for hemophilia B.
Accordingly, it is an object of the present invention to provide a method for producing a transgenic animal that secretes biologically human active Factor IX into its milk.
It is a further object of this invention to provide a transgenic animal that produces at least 100 xcexcg of human Factor IX per milliliter of milk.
These and other objects are achieved, in accordance with one embodiment of the present invention by the provision of a transgenic non-human mammal containing an exogenous DNA molecule stably integrated in its genome. A non-human transgenic mammal containing an exogenous DNA molecule stably integrated in its genome, wherein said exogenous DNA molecule comprises:
(a) 5xe2x80x2 regulatory sequences of a mammary gland-specific gene including a promoter;
(b) a Factor IX-encoding DNA sequence that encodes a signal sequence, a Factor IX pro-sequence and a Factor IX sequence in a 5xe2x80x2to 3xe2x80x2 direction, wherein said signal sequence is effective in directing the secretion of said Factor IX into the milk of said transgenic mammal and wherein said Factor IX sequence lacks at least a portion of the complete or the complete 5xe2x80x2-untranslated and 3xe2x80x2-untranslated regions of the Factor IX gene.; and
(c) 3xe2x80x2 regulatory sequences from a mammary gland-specific gene or 3xe2x80x2 regulatory sequences active in a mammary gland;
wherein said 5xe2x80x2 and said 3xe2x80x2 regulatory sequences are operatively linked to said Factor IX-encoding DNA sequence.
Mammary gland-specific promoters that are useful in the present invention are selected from the group consisting of short whey acidic protein (WAP) promoter, long WAP promoter, short xcex1-casein promoter, short xcex2-casein promoter, short kappa-casein promoter, long xcex1-casein promoter, long xcex2-casein promoter, long kappa-casein promoter, xcex1-lactalbumin promoter and xcex2-lactoglobulin promoter.
Non-human transgenic mammals which are contemplated by the present invention are selected from the group consisting of mice, rats, rabbits, pigs, sheep, goats and cows.
It is a further object to provide a process for producing Factor IX by providing a non-human transgenic mammal having integrated into its genome an exogenous DNA molecule, wherein said exogenous DNA molecule comprises:
(a) providing a non-human transgenic mammal having integrated into its genome an exogenous DNA molecule, wherein said exogenous DNA molecule comprises: (1) 5xe2x80x2 regulatory sequences of a mammary gland-specific gene including a promoter; (2) a Factor IX-encoding DNA sequence that encodes a signal sequence, a Factor IX pro-sequence and a Factor IX sequence in a 5xe2x80x2 to 3xe2x80x2 direction, wherein said signal sequence is effective in directing the secretion of said Factor IX into the milk of said transgenic mammal and wherein said Factor IX sequence lacks at least a portion of the complete or the complete 5xe2x80x2-untranslated and 3xe2x80x2-untranslated regions of the Factor IX gene.; and (3) 3xe2x80x2 regulatory sequences from a mammary gland-specific gene or 3xe2x80x2 regulatory sequences active in a mammary gland; wherein said 5xe2x80x2 and said 3xe2x80x2 regulatory sequences are operatively linked to said Factor IX-encoding DNA sequence;
(b) allowing said DNA sequences encoding said Factor IX to be expressed and said Factor IX to be secreted into the milk of said transgenic mammal;
(c) collecting said milk from said mammal; and
(d) isolating said Factor IX from said milk.
It is a further object to provide a method of treating hemophilia B using the Factor IX produced by the transgenic mammal, described above. Treating involves administration of the Factor IX of the invention and a pharmaceutically acceptable carrier to a hemophilia B patient.
It is a further object of the invention to provide an in vitro culture of mammary gland cells that produce Factor IX. Another object of the invention is to provide a method of treating hemophilia B by implanting such Factor IX mammary gland cells into a patient.